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BIOLOGICAL CONTROL

Effects of Different Formulations on Viability and Medium-Term Storage of Metarhizium anisopliae Conidia ROBERTO T. ALVES1, ROY P. BATEMAN2, JANE GUNN2, CHRIS PRIOR3 AND SIMON R. LEATHER4 1

EMBRAPA Cerrados, Rodovia Brasília-Fortaleza BR 020, km 18, 73301-970, Planaltina, DF 2 CABI Bioscience, Silwood Park, Buckhurst Road, Ascot, Berks, SL5 7TA, UK 3 The Royal Horticultural Society, Wisley, Woking, Surrey, GU23 6QB, UK 4 Silwood Centre for Pest Management, Dept. Biology, Imperial College, Silwood Park, Buckhurst Road, Ascot, Berks, SL5 7PY, UK Neotropical Entomology 31(1): 091-099 (2002)

Efeitos de Diferentes Formulações na Viabilidade e no Armazenamento de Conídios de Metarhizium anisopliae a Médio Prazo RESUMO – O objetivo deste trabalho foi avaliar os efeitos de diferentes formulações na viabilidade de conídios de Metarhizium anisopliae. No experimento 1, a viabilidade de conídios misturados com oito óleos adjuvantes emulsionáveis (OAE), sete espalhantes adesivos, três óleos vegetais e quatro óleos minerais foi avaliada 24h e 48h depois de espalhados sobre o meio de SDA. Formulações que não apresentaram nenhum efeito adverso sobre a viabilidade dos conídios foram utilizadas no experimento sobre armazenamento. No experimento 2, avaliaram-se os efeitos do armazenamento a 10ºC e 27ºC durante 40 semanas de conídios de M. anisopliae var. acridum formulados em cinco OAE, em um óleo vegetal e em uma mistura de dois óleos minerais, além de conídios puros e secos. No experimento 1, as formulações em óleo não causaram nenhum efeito negativo na germinação dos conídios. Os tratamentos com os óleos adjuvantes Actipron, Ashlade e Codacide proporcionaram níveis de germinação semelhantes aos obtidos com óleos minerais e vegetais, 24h após a incubação. O espalhante Ethoken C12 foi letal para os conídios. No experimento 2, a viabilidade conidial de uma mesma formulação declinou com o passar do tempo. A viabilidade foi mantida acima de 90% a 10ºC em todas os formulantes testados. A formulação com Ashlade (OAE) proporcionou níveis de germinação semelhantes aos obtidos com a mistura Shellsol e Ondina, a 10ºC e 27ºC durante 40 semanas. Logo, OAE podem ser usados para formular conídios de M. anisopliae sem nenhum efeito adverso na viabilidade dos mesmos. PALAVRAS-CHAVE: Óleo adjuvante, formulação em óleo emulsionável, micoinseticida. ABSTRACT – The aim of this work was to evaluate the effects of different formulations on the viability of Metarhizium anisopliae conidia. In a first experiment, the viability of conidia mixed with eight emulsifiable adjuvant oils (EAO), seven wetter/spreaders, three vegetable oils and four mineral oils was evaluated 24h and 48h after spreading over SDA medium surface. Some formulations, which did not present any adverse effect on conidial viability in the first 24h of incubation, were recommended for the medium-term storage experiment. In a second experiment, the effects of different formulations on medium-term storage (40 weeks) of M. anisopliae var. acridum conidia were evaluated at 10ºC and 27ºC. Five EAO, one vegetable oil, a mixture of mineral oils and pure dry conidia were tested. In the experiment 1, the oil formulations did not cause any negative effect on conidial germination. The treatments with the adjuvant oils Actipron, Ashlade and Codacide gave germination levels equal to mineral and vegetable oils after 24h of incubation. The wetter/spreader Ethoken C12 was lethal to conidia. All tested emulsifiable adjuvant oil-based formulations were compatible to the conidia as the oil-based formulations after 48h of incubation. In the experiment 2, conidial viability within the same formulation declined over time. Conidial viability was maintained above 90% at 10ºC in all tested formulants. The conidial suspension with Ashlade gave equal germination levels to Shellsol plus Ondina, when stored at 10ºC and 27ºC for 40 weeks. Thus EAO can be used to formulate M. anisopliae conidia without adverse effects on viability. KEY WORDS: Adjuvant oil, emulsifiable oil-based formulation, mycoinsecticide.

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The development of a suitable formulation is essential to the successful utilisation of commercial mycoinsecticides (Daoust et al. 1983). For example, many formulations can affect the conidial viability resulting in a short shelf life (Moore & Prior 1993). There is a need for careful assessment of the compatibility of formulation components with conidia prior to their use in formulations (Daoust et al. 1983). Therefore, one of the first steps in developing a mycoinsecticide formulation is to evaluate the effects of its components on conidial viability to select products compatible with fungal conidia. Formulating pathogens in oil enhances their infectivity compared to conventional water-based formulations (Agudelo & Falcon 1983, Prior et al. 1988, Bateman et al. 1993). After that, studies on medium and long-term storage of the entomopathogenic fungi Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) formulated in mineral or vegetable oils are usually carried out (Stathers et al. 1993, McClatchie et al. 1994, Hedgecock et al. 1995, Moore et al. 1995, Moore et al. 1996). These works were mainly carried out using M. anisopliae var. acridum, previously known as M. flavoviride Gams and Rozsypal. Views can differ on how long a mycoinsecticide shelf life is required, but estimates range from three to 18 months or even longer (Moore & Prior 1993). The same authors say that it is desirable to maintain the viability to cover two cropping seasons, and long term storage is more for the convenience of the manufacturers than of the farmer and should not be allowed to remain an obstacle. In general, temperature and moisture content, or the humidity of the storage atmosphere are the major factors which influence conidial longevity (Hong et al. 1997). Hedgecock et al. (1995) studied the influence of moisture content on temperature tolerance and storage of M. anisopliae var. acridum in oil formulation and the results demonstrated that viability declined due to high temperatures and high moisture contents. Drying the conidia with silica gel greatly improved high temperature tolerance (McClatchie et al. 1994). The optimal moisture content for dried conidia storage was found to be 4-5% and a range of mineral oils proved satisfactory for dried conidia storage (Moore et al. 1996). Less moisture content than 4-5% may give better results but it is difficult to achieve. Suspoemulsions can be defined as heterogeneous formulations consisting of a stable dispersion of active ingredients in the form of solid particles and of fine globules in a continuous water phase combinations (GCPF 1994). They are relatively new to the agricultural market and have a great potential for formulation and application of mycoinsecticides for pest control. They can be sprayed by very low volume/ controlled droplet application techniques and still allow the use of conventional hydraulic sprayers and nozzles and water - the cheapest and most readily available carrier liquid for pesticides (Alves et al. 1998). A suspoemulsion, containing an emulsifiable adjuvant oil plus conidia of M. anisopliae and water, was as infective as oil-based fungal formulations and more infective than conventional water-based fungal formulations against the yellow mealworm, Tenebrio molitor, larvae (Alves et al. 1998). Little information is available about the effects of different

commercially available products, which could be used in water-based formulations, on conidial viability and shelf life. Hence, further studies are required. The aims of this investigation were: 1) to evaluate the effects of different formulations on the viability of M. anisopliae conidia after 24h and 48h incubation. Some formulations which do not present any adverse effect on conidial viability in the first 24h of incubation, will be recommended for the mediumterm storage experiment; 2) to evaluate the effects of a range of commercially available formulations on medium-term storage (40 weeks or 9.3 months) of M. anisopliae var. acridum conidia, at two different temperatures. Results on emulsifiable adjuvant oil-based conidial formulations were emphasised in this work, because they have a great potential to be sprayed in broad scale agriculture for pest control, where water-based formulations are predominant.

Material and Methods Experiment 1. Effects of Different Formulations on M. anisopliae Conidial Viability. Conidia of M. anisopliae isolate 299984 originally obtained from sugarcane froghopper, Aeneolamia varia saccharina (Homoptera: Cercopidae), in Trinidad, were used in this experiment because they were highly virulent against the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae), a model insect used in previous bioassays (Alves unpublished). Aerial conidia were grown on Sabouraud-Dextrose-Agar medium (SDA) in a petri dish of 5 cm diameter and 1 cm deep at 25±0.5ºC under darkness. After 10 days, conidia were harvested using a spatula and then suspended in a total volume of 10 ml of 22 different formulations. Conidia were formulated in different emulsifiable adjuvant oil, wetter/spreader and oil formulations for viability comparison. The water-based and oil-based formulations tested in this study are presented in Tables 1 and 2, respectively. Conidia were mixed with emulsifiable adjuvant oils and wetter/spreader agents prior to the addition of distilled water to obtain homogeneous suspensions. The stock formulation of each replicate was filtered using a sterilized muslin cloth then mixed using a Whirli Mixer (FSA Laboratory, U.K.) for 3 min. to break down conidial chains and to reduce clumping. All conidial formulations were then calibrated at a concentration of 10 6 conidia ml -1 using an improved Neubauer’s chamber. The resultant formulations remained resting for 2h, to allow the conidia to be sensitised to any adverse effect caused by the formulants. After that, they were thoroughly agitated for 10 seconds using the Whirli Mixer. Aliquots of 0.1 ml from each formulation were then pipetted by an Eppendorf Research piston-stroke pipette (EppendorfNethler, Germany) and thinly spread over the SDA surface in a 5 cm diameter petri dish 1cm deep. The plates were incubated at 25±0.5ºC. Conidial viability tests were carried out after 24h and 48 h of incubation to allow time for conidia recovery from any adverse effect caused by the formulations. Conidia were examined at 400x magnification and germination was recorded when the germ tube was visible. All the conidia in each field of view were counted to obtain at least a total of

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Table 1. Water-based formulations, compositions and suppliers. Formulation

Composition

10% Actipron

Adjuvant oil containing 97% highly refined mineral oil plus emulsifiers Adjuvant oil containing 99% highly refined paraffinic oil plus emulsifiers Adjuvant oil containing 95% rapeseed oil and 5% emulsifiers Adjuvant oil containing 99% highly refined paraffinic oil and 1% surfactant Adjuvant oil containing 95% emulsifiable rapeseed oil plus emulsifiers Adjuvant oil containing a blend of refined vegetable and emulsifying agent Adjuvant oil containing 94% pure rapeseed oil plus emulsifiers Adjuvant oil containing 60% mineral oil and 40% surfactants Laboratory wetting agent made of polyoxyethylenesorbitan monooleate Non-ionic wetter/spreader containing 948 g/l alkyl phenol ethylene oxide Non-ionic wetter/spreader containing 900 g/l phenol ethylene oxide condensate Cationic surfactant containing 870 g/l polyoxyethylene tallow amine Cationic surfactant containing bis-2 hydroxyethyl coco-amine

10% Ashlade 10% Codacide 10% Cropspray 11E 10% Cutinol 10% Emoleo R2 10% Natur’l oil 10% Output 0.05% Tween 80 0.1% Agral 0.1% Enhance 0.1% Ethoken 0.1% Ethoken C12

Supplier Bayer Ag., Germany Ashlade Formulations Ltd., U.K. Microcide Ltd., U.K. Newman Agrochemicals Ltd., U.K. Techsol Ltd., U.K. John L. Seaton & Co. Ltd., U.K. Stoller Chemical Ltd., U.K. Zeneca Crop Protection, U.K. Sigma Chemicals, U.K. Zeneca Crop Protection, U.K. Techsol Ltd., U.K. Techsol Ltd., U.K. Techsol Ltd., U.K.

0.1% Silwet L77

Wetter organosilicone co-polymer containing 100% active liquid with a minimum of 80% polyalkylene oxide modified heptamethyltrisiloxane and a minimum of 20% alkyloxypolyethylene glycol methyl ether

Newman Agrochemicals Ltd., U.K.

0.1% Spreader

Non-ionic wetter/spreader containing nonylphenol ethylene oxide condensate

Pan Britannica Industries Ltd., U.K.

Table 2. Oil-based formulations, compositions and suppliers. Formulation

Composition

Supplier

Shellsol T

Refined paraffinic oil

Alcohols Ltd., U.K.

Ondina EL


Shell Oil Co., U.K.

50% Shellsol T + 50% Ondina EL

Mixture of refined paraffinic oils

Alcohols Ltd. and Shell Oil Co., U.K.

Isopar M


Exxon Company, U.S.A.

Peanut oil

Vegetable oil with high viscosity and low volatility

Sigma Chemicals, U.K.

Soyabean oil



Sunflower oil



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300 conidia in a range between 300 and 400, for each replicate (Moore et al. 1993). The experiment had a factorial design with two main factors (formulations with twenty-two levels and incubation time with two levels) and three replicates. Factorial analysis of variance (ANOVA) on conidial viability data was performed using the statistical package SPSS for Windows (Norusis & SPSS 1993), after transforming the data to Arcsin √ (%/100) to meet the requirements of ANOVA for a normal data distribution and homogeneity of variances. The results are presented untransformed in tables. Experiment 2. Effects of Different Formulations on Medium-term Storage of M. anisopliae var. acridum Conidia. The isolate IMI 330189 of M. anisopliae var. acridum, originally isolated from a grasshopper Ornithacris cavroisi (Orthoptera: Acrididae) in Niger, was used in this experiment because it has been applied in several bioassays and field trials against locusts and grasshoppers in Africa and Australia (Lomer et al. 1993, Bateman et al. 1994, Milner et al. 1994, Price et al. 1997). This isolate is commercially available in the South African market (Bateman et al. 1998). Aerial conidia were produced using a standard two-phase production system (Jenkins et al. 1998), which consists of a submerged liquid culture followed by conidiation on a solid substrate. The submerged liquid culture uses a simple liquid medium containing 20 g l-1 of dried yeast, 20 g l-1 of glucose and 1000 ml of tap water and generates hyphal bodies and mycelium for inoculation into the second phase. Autoclaved broken white rice in polypropylene bags was used as solid substrate in the second phase. The bags were incubated for 12 days at 25±1ºC for fungal development and sporulation. The plastic bags were then opened, the rice with conidia was spread out in clean plastic trays and placed inside a Fisons drying cabinet (Fisons Scientific Equipments, U.K.) at 20±3ºC. After five days, the moisture content was around 20%. Conidia were then extracted from the rice by sieving through brass laboratory sieves (300 mm mesh). After sieving, conidia were dried further in an auto-desiccator cabinet with a built-in hygrometer (Whatman International Ltd., U.K.) for four days to reduce the moisture content to around 5%, because a suitable optimal moisture content for long term dried conidia storage was found to be 4-5% (Moore et al. 1996). The initial moisture content of six randomised 0.5 g samples of conidia, from the same batch which was used in the experiment, was assessed by a Mettler-Toledo HG53 Halogen Moisture Content Analyser (Mettler-Toledo, Switzerland). The analyser detected a mean moisture content of 4.93%. One gram of conidia was weighed and mixed with 200 ml of distilled water plus 0.05% Tween 80 using a Whirli Mixer for 3 min. to break down conidial chains and to reduce clumping. The number of conidia/ml was counted using an improved Neubauer’s chamber and the number of conidia g1 was calculated. This procedure was repeated six times. The mean result was 4.2 x 1010 conidia g-1. The appropriate number of grams of conidia to be added to the formulants and to facilitate the germination countings later, was calculated. Then, 0.01 g of pure conidia containing approximately 4.2 x

108 conidia were mixed with 15 ml of eight formulations selected from the previous experiment (with conidial viability above 90% after 24h of incubation), resulting in a suspension with approximately 2.8 x 107 conidia ml-1. The formulations were the following: pure Ashlade, pure Codacide, pure Cropspray 11E, pure Emoleo R2, pure Natur’l oil, pure peanut oil and in a mixture of Shellsol T plus Ondina EL. There was also a treatment with 0.8 g of pure dry conidia (technical material) as a standard for general comparisons. Fresh dry non-indicating silica gel beads (20%) were added to 15 ml of each formulation and to the pure dry conidia treatment, to absorb any remaining moisture content from the formulations and to maintain the moisture content between 4 and 5% (Moore et al. 1996). The 20 ml Universal bottles were sealed with parafilm and stored at 10 ºC and 27ºC. Viability of stored conidia from different formulations was assessed one day after formulation to obtain the initial germination level and at intervals of five weeks for 40 weeks. To facilitate the germination counting, 0.1 ml aliquots (containing approximately 2.8 x 106 conidia) from each stored conidial suspension were pipetted by an Eppendorf pipette and mixed with 5 ml of distilled water for the adjuvant oil treatments, 5 ml of pure Shellsol T for the peanut oil and for Shellsol plus Ondina treatments (resulting in a suspension with approximately 5.6 x 105 conidia ml-1). For the pure dry conidia treatment, a small amount of conidia weighing approximately 0.007 g was mixed with 5 ml of water plus 0.05% Tween 80 treatments (resulting in a suspension with approximately 5.88 x 107 conidia ml-1). It was diluted 100 times to obtain approximately 5.88 x 105 conidia ml-1. All resultant diluted conidial formulations were then mixed using a Whirli Mixer for 3 min. to homogenize the suspensions. Finally, a new aliquot of 0.1 ml (containing approximately 5.6 x 104 conidia) was pipetted from each treatment and thinly spread over the SDA surface in a 5 cm diameter petri dish 1cm deep. The plates were incubated at 25±0.5ºC. Conidial viability tests were carried out after 24h of incubation using the same methodology of the previous experiment (Moore et al. 1993). To check that the moisture content after 40 weeks from the three replicates with pure dry conidia stored at the two different temperatures was similar to the initial values, new assessments were carried out using the same equipment and methodology. This experiment had a factorial design with three main factors (formulations with eight levels, temperature with two levels and storage time with nine levels) and three replicates. A three-way ANOVA on conidial viability data was performed using the same statistical package used in the previous experiment. The data were transformed to Arcsin √ (%/100) to meet the requirements of ANOVA for a normal data distribution and homogeneity of variances.

Results Experiment 1. Effects of Different Formulations on M. anisopliae Conidial Viability. There were significant differences between formulations on conidial viability (df = 21, 131, F = 278.31, P